The role of calcium in contraction and relaxation of the myofibril is well established. The mechanisms by which the smooth muscle cell regulates calcium levels are poorly documented. The long term goal is an understanding of the control mechanism for intracellular homeostasis. This proposal contains experiments to verify the following hypothesized reaction sequence in bovine uterine smooth muscle microsomes. (1) E + Ca degrees + ATP degrees = EATPCa degrees (2) EATPCa degrees = E*PCai + ADP degrees (3) E*PCai + Mgi = E*PMgi + Cai (4) E*PMgi = Pi degrees + E + Mg degrees Our goal is to demonstrate for the first time in smooth muscle several of these reactions. Specifically, we will demonstrate phosphorylation of a protein and its correlation with a Ca, Mg-ATPase (equation 1), both by activity, inhibition of enzyme activity, and electrophoretic studies. Furthermore, the reversibility of reactions 1 and 2 will be documented by ATP formation from ADP, coupled with calcium efflux. Optimal conditions for each of these reactions will be worked out. Equation 4 and 3 will be substantiated by showing the phosphorylation of the calcium transport ATPase from inorganic phosphate and magnesium, and the translocation of magnesium. We will also document the full reverse reaction sequence 4-1, driven by a calcium gradient and addition of magnesium, Pi, and ADP. In addition, we will attempt to distinguish between intravesicular and surface membrane bound calcium. Studies on uterine muscle will enhance our understanding of abnormal uterine contractility and serve to develop therapy for premature labor and dystocic labor.